Various publications, including patents, published applications, accession numbers, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference, in its entirety and for all purposes, in this document.
Classical methods of vaccine production use either modified live (e.g., attenuated) virus or killed (e.g., inactivated) virus to stimulate immunity. The development and accelerated production of safer and more stable vaccines is desirable for combatting highly virulent pathogens of interest. In addition, vaccines that are thermo-stable are needed for use and distribution in areas of the world where refrigeration is scarce and supplies must be stored in unregulated conditions where temperatures are well in excess of that required to maintain the potency of typical vaccine formulations. An example of the urgent need for such thermo-stable vaccines has been the Ebola outbreak in towns and remote villages of Africa.
Ebola virus (EBOV), a member of the order of enveloped viruses Mononegavirales, causes a severe hemorrhagic disease in humans, exhibiting mortality rates as high as 90%.1 This high mortality rate, combined with an absence of efficacious treatments or established vaccination options, makes Ebola virus a critical public health pathogen, and a bio threat pathogen of category A.
The EBOV surface glycoprotein (GP) mediates attachment to and fusion of the virus with host cells. GP is a type 1 transmembrane protein that is displayed on the virion envelope as a homo-trimeric spike, similar in fashion to several other envelope antigens (e.g., HIV Env and influenza virus hemagglutinin (HA)). The GP protein is exposed on the surface of Ebola virions, and it is widely recognized as the primary antigen of Ebola—thus vaccination with a purified, native-like but noninfectious protein is an obvious choice for an effective vaccination strategy.
The extracellular domain of the Zaire Ebola virus (ZEBOV) GP protein, lacking its transmembrane domain, has been fused to the Fc fragment of human IgG1 and the resultant fusion protein was successfully expressed in mammalian cells. Although this sort of soluble construct conferred protection to mice against a lethal viral challenge, it failed when similarly tested in non-human primates (NHP). Vaccines that utilize soluble forms of virion envelope antigens may not properly mimic the native structure of the membrane-integrated glycoprotein displayed on the virion for the purpose of immunization. This perspective is supported by studies in which antigens expressed in their native conformation are more effective at eliciting an effective immune response than in their non-native form. Indeed, virus-like particles (VLP) that present full-length, native GP on their surfaces have recently been demonstrated to be efficacious in eliciting protective immunity in NHP.
Although VLPs can be produced by transfecting mammalian cell lines with the EBOV matrix protein (VP40) and GP, VLPs are less than ideal vaccines for a number of reasons. Primarily, they are particulate in nature, and thus impossible to separate from other particulate contaminants like adventitious viruses. Because they have enclosed spaces it is exceedingly difficult to ensure the complete removal or inactivation of endogenous host nucleic acids or entrapped host proteins. Since the envelopes of VLPs are derived from the membrane of the host cell that produces them, they consequently contain many additional membrane-anchored or membrane-integrated proteins originating from the host cell membrane. Yet the success of using Ebola VLPs to elicit effective immune responses in NHP suggests that if envelope antigens could be highly purified and delivered in a vaccine in their native, membrane-embedded form, just as they are in VLPs, they may be equally effective in promoting protective immunity.
If envelope antigens could be delivered in a vehicle with built-in adjuvant properties, they might even prove more effective, and without the drawbacks of VLPs with respect to contamination with host-derived or potentially infectious, adventitious contaminants.